Injury to axons of the central nervous system, whether in the form of a physical injury or neurodegenerative diseases, remains a significant burden in modern medicine. Whereas axons of the peripheral nervous system readily regenerate and innervate targets, those of the central nervous system do not. Therefore, enabling axon regeneration represents a crucial hurdle in regenerative medicine. The insulin-signaling and AKT/mTOR pathways are evolutionarily conserved mechanisms that promote axon regeneration. Utilizing a powerful model of advanced laser surgery to perform axotomy of single neurons within adult Caenorhabditis elegans, I have identified O-linked N-beta-acetylglucosamine (O-GlcNAc) post-translational modifications as a novel regulator of axon regeneration. Loss-of-function mutations in the O-GlcNAc Transferase, the enzyme that adds O- GlcNAc to target protein serines/threonines, significantly enhances axon regeneration. Astonishingly, loss-of- function mutations in the O-GlcNAcase, the enzyme that removes O-GlcNAc, also enhances regeneration. This counterintuitive result suggests that these enzymes act in independent pathways to regulate regeneration. The aims of this proposal are to: 1. Determine how decreased O-GlcNAcylation in neurons increases axon regeneration through the AKT/mTOR pathway. 2. Determine how increasing O-GlcNAcylation enhances regeneration through the insulin-signaling pathway. This will be done by utilizing the power of C. elegans genetics and mouse neuronal cultures to pinpoint where and how O-GlcNAcylation is acting. Pharmacological analysis will further define the role of O-GlcNAcylation and identify potential therapeutic targets. This proposal will significantly advance the field of axon regeneration by defining a novel cellular mechanism where by O- GlcNAc modifications act as critical modulators of axon regeneration.